Use of selective gaba a alpha 5 negative allosteric modulators for the treatment of central nervous system conditions

ABSTRACT

The present invention relates to the pharmaceutical use of selective GABA A α5 negative allosteric modulators for the treatment, prevention and/or delay of progression of central nervous system (CNS) conditions related to excessive GABAergic inhibition in the brain.

PRIORITY TO RELATED APPLICATION(S)

This application claims the benefit of European Patent Application No.10190267.4, filed Nov. 5, 2010 and European Patent Application No.10191396.0, filed Nov. 16, 2010, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Down syndrome (DS), caused by triplication of chromosome 21, is the mostfrequent genetic cause of intellectual disability, with a prevalence ofabout one in 650-1000 live births worldwide [Bittles A H et al., Eur JPublic Health (2007) 17(2): 221-225]. Even though the etiology of thecognitive deficit in DS remains uncertain, cellular and anatomicalabnormalities in the prenatal and perinatal forebrain and cerebellumsuggest that early brain development is altered in individuals with DS.Similar central nervous system (CNS) abnormalities have been describedin mouse models of DS. In particular, the Ts65Dn mouse, the most widelyused model of DS, has abnormal forebrain and cerebellar development,defects in synapse formation and neurophysiology, and behavioraldeficits.

Recent studies have suggested that the major functional defect in thepostnatal Ts65Dn brain may be an imbalance between excitation andinhibition, e.g. a decreased numbers of excitatory synapses and arelative increase in inhibitory synaptic markers in the cortex andhippocampus. Further studies suggest that increased inhibitory synapticdrive may be a general physiological phenotype in the Ts65Dn forebrain.

There is currently no therapeutic option available for the treatment ofcognitive deficit in people with DS. It has now been found, thatinhibition of GABA A receptor function represents an attractivemechanism to treat cognitive impairment in DS.

The GABA A receptor regulating a chloride channel is the predominantinhibitory neurotransmitter receptor in the mammalian central nervoussystem and has been widely used as a target for neuromodulatory drugs.Many compounds in clinical use such as anxiolytics, sedatives, hypnoticsor anti-epileptics increase GABA A receptor activation via theallosteric benzodiazepine (BZD) binding site. Such compounds have beentermed “BZD site receptor agonists.” BZD binding site ligands producingthe opposite effect, i.e., decreasing receptor activation, are called“BZD site receptor inverse agonists.” “BZD site receptor antagonists”are ligands which bind to the receptor without modulating its functionbut which block the activity of both agonists and inverse agonists[Haefely W E, Eur Arch Psychiatry Neurol Sci (1989) 238: 294-301]. BZDreceptor inverse agonists have so far only been tested in animalbehavior experiments and in a very few exploratory human studies. Theresults showed beneficial activity, however, further development of thecompounds that entered the clinic was prevented by anxiogenic effects,possibly resulting from the lack of selectivity shown by these agentsfor specific BZD receptor subtypes.

Non-selective antagonists, also called channel blockers, of the GABA Areceptors (e.g. picrotoxin or PTZ) increase the risk of convulsions mostlikely through their actions on GABA A α1, α2, and α3 subunit containingreceptors and, therefore, cannot be safely used in people with DS. It ishence a prerequisite that suitable GABA A receptor inhibitors areselective for the receptor subtype mainly involved in memory formation.

GABA A receptors are pentamers mostly consisting of two α, two β and a γsubunit. Several gene products are available for each of the subunitsgiving rise to a large number of receptor variants. The importance ofdifferent a subunit subtypes has been elucidated by the generation oftransgenic mice lacking the normal diazepam sensitivity of the α1, α2,α3 or α5 subunit (α4 and α6 are diazepam insensitive). The resultssuggest that α1 is responsible for the sedative effects and α2 andperhaps α3 for the anxiolytic effects of BZD receptor ligand agonists[Löw K et al., Science (2000) 290(5489): 131-134; Möhler H, Cell TissueRes (2006) 326(2):505-5161. The consequences of a modified pharmacologyof the α5 subunit are less evident, but reduced or no expression of thesubunit could be associated with facilitated cognition inhippocampal-dependent tasks and importantly, no effects on anxiety orproconvulsant paradigms. This is in line with the preferentiallocalization of α5 subunits in the hippocampus.

SUMMARY OF THE INVENTION

The present invention provides methods for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsrelated to excessive GABAergic inhibition in the cortex and hippocampuswhich comprises administering selective GABA A α5 negative allostericmodulators. More particularly, the present invention provides methodsfor the treatment, prevention and/or delay of progression of CNSconditions caused by neurodevelopmental defects which result inexcessive GABAergic inhibition in the cortex and hippocampus, whereinthe CNS condition is selected from cognitive deficits in Down Syndrome,in autism, in neurofibromatosis type I, or for the recovery after strokewhich comprises administering selective GABA A α5 negative allostericmodulators.

In particular, the present invention provides methods for the treatment,prevention and/or delay of progression of CNS conditions, whichcomprises administering selective GABA A α5 negative allostericmodulators wherein the selective GABA A α5 negative allosteric modulatoris a compound of formula (I) and/or a compound of formula (II)

wherein R¹, R², R³, R⁴, R⁵, and R⁶ are as defined herein, or apharmaceutically acceptable salt thereof.

It is therefore hypothesized that a BZD site ligand with inverse agonismselective for GABA A α5 subunit-containing receptors should enhancecognitive function without anxiogenic and proconvulsant side effects.

Selectivity of a BZD site ligand can be achieved by different affinitiesto GABA A receptor subtypes (“binding selectivity”). Alternatively, inthe case of similar subtype affinities, different degrees of receptormodulation (“functional selectivity”) can be attempted, i.e., inverseagonism at GABA A α5 receptor subtype and no activity at other subtypes.A compound may also have a combination of both binding and functionalselectivity although so far this is rare. A number of compoundsdescribed as being active as inverse agonists at the GABA A α5subunit-containing receptors have recently been synthesized [WO2006/045429, WO 2006/045430, WO 2007/042421, WO 2009/071476]. Certain ofthese compounds have a beneficial pharmacological profile with excellentbinding and functional selectivity for the GABA A α5 subunit-containingreceptors. Results confirm the hypothesis that compounds with such apharmacological profile can improve cognitive function withoutCNS-mediated adverse effects including anxiety and/or convulsions[Ballard T M et al., Psychopharmacology, (2009) 202: 207-223].

The pharmaceutically active compounds used in present invention aremolecules combining both binding and functional selectivity at the GABAA α5 subunit-containing receptors that improve cognition. Importantly,pharmaceutically active compounds used in present invention lackanxiogenic or proconvulsant effects at the exposures tested intoxicology studies.

Selective GABA A α5 negative allosteric modulators have procognitiveeffects on several animal models but are not anxiogenic orproconvulsant. The active pharmaceutical compounds used in presentinvention were chronically administered to Ts65Dn and control (euploid)mice and a battery of behavioral tests, including the assessment ofsensorimotor abilities, anxiety and cognition was performed. The activepharmaceutical compounds used in present invention improved Ts65Dn, butnot control, mice performance in the Morris water maze and did notaffect the sensorimotor abilities, general activity, motor coordinationor anxiety of Ts65Dn or control mice. Plasma concentrations of activepharmaceutical compounds from blood samples taken from treated Ts65Dnand control mice relate to levels of GABA A α5 receptor occupancy of25-75% from an in vivo binding mimic study. Importantly, theseexperiments confirmed the selective occupancy of brain GABA A α5receptors and reinforce the notion that dual binding and functionalselectivity offers an ideal profile for cognition-enhancing effectswithout the unwanted side effects associated with activity at other GABAA receptor subtypes

Interestingly it was revealed that chronic administration of the activepharmaceutical compounds used in present invention:

1. did not modify any of the sensorimotor abilities tested in Ts65Dn orcontrol mice;2. did not affect motor coordination in the Rotarod test;3. did not modify spontaneous locomotor activity in the home-cage duringthe light or the dark phase of the cycle;4. in the Open Field test did not modify the anxiety or locomotoractivity of Ts65Dn and control mice;5. in the Hole Board test reduced the hyperactivity found invehicle-treated Ts65Dn mice;6. in the Morris water maze it improved Ts65Dn mice performance duringthe acquisition and the cued sessions.

It was further found that the active pharmaceutical compounds used inpresent invention:

a) reverse the spatial learning deficits in Nf1+/− mutant mice underconditions in which such compounds do not enhance learning in controlmice;b) do not affect the performance of Nf1+/− mice under conditions thatocclude their behavioral deficits;c) do not affect motor learning in the rotarod test in Nf1+/− andcontrol mice;d) are useful as potential treatments for the cognitive deficitsassociated with NF1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Mean±S.E.M. of the latency of 8581-, R1- and vehicle-treatedTs65Dn and control mice to fall from the rotarod at different constantspeeds.

FIG. 2. Mean±S.E.M. of the latency of 8581-, R1- and vehicle-treatedTs65Dn and control mice to fall from the rotarod during the accelerationcycle.

FIG. 3. Mean±S.E.M. of the spontaneous activity performed by Ts65Dn andcontrol mice under vehicle R1 or 8581 treatment in their home-cageacross a complete dark-light cycle of 24 hours.

FIG. 4. Mean±S.E.M. of the mean activity performed by Ts65Dn and controlmice under vehicle, R1 or 8581 treatment in the light and the dark phaseof the cycle.

FIG. 5. Mean±S.E.M. of the number of crossings performed by R1-, 8581-and vehicle-treated Ts65Dn and control mice in the center and peripheryof the open field. *: p<0.05; **: p<0.01 Bonferroni tests aftersignificant ANOVA.

FIG. 6. Mean±S.E.M. of the number of rearings performed by R1-8581- andvehicle-treated Ts65Dn and control mice in the open field.

FIG. 7. Mean±S.E.M. of the latency to reach the platform during theeight acquisition sessions in the MWM.

FIG. 8. Mean±S.E.M. of the latency to reach the platform during theeight acquisition sessions by Ts65Dn and control vehicle-(A) 8581-(B)and R1-treated mice and by vehicle and 8581-treated Ts65Dn (C) andcontrol (D) mice. *:p<0.05; **: p<0.01; ***: p<0.001 T-test aftersignificant ANOVAs.

FIG. 9. Mean±S.E.M. of the latency to reach the platform during theeight acquisition sessions by vehicle and 8581-treated Ts65Dn (A) andcontrol (B) mice and by vehicle and R1-treated Ts65Dn (C) and control(D) mice. *:p<0.05; **: p<0.01; ***: p<0.001 t-test after significantANOVAs.

FIG. 10. Mean±S.E.M. of the latency to reach the platform during thecued sessions. *: p<0.05 Ts65Dn vs. control; #: p<0.05; ##: p<0.01 8581and R1 vs. vehicle.

FIG. 11. 8581 reverses the deficit in Long-term potentiation inhippocampal slices of Ts65Dn mice after chronic treatment. Data arepresented as means±S.E.M. of evoked EPSP recorded from hippocampalslices of vehicle, 8581 treated Ts65Dn (TS) and control (CO) mice. Aftera 20 min stable baseline period, tetanic stimulation was applied tohippocampal slices to induce LTP. Field ESPS slopes were normalized andpresented as mean±SEM (n=5-7/group).* p<0.05 vs. vehicle (V).

FIG. 12. R1 reverses the deficit in Long-term potentiation inhippocampal slices of Ts65Dn mice after chronic treatment. Data arepresented as means±S.E.M. of evoked EPSP recorded from hippocampalslices of vehicle, R1 treated TS and CO mice. After a 20 min stablebaseline period, tetanic stimulation was applied to hippocampal slicesto induce LTP. Field ESPS slopes were normalized and presented asmean±SEM (n=5-7/group).* p<0.05 vs. vehicle (V).

FIG. 13. 8581 rescues neuronal proliferation in the hippocampus of TSand CO mice. Data are expressed as means±S.E.M. of the density of Ki67+cells in vehicle- and 8581-treated TS and CO mice. ANOVA ‘genotype’:F(1, 20)=7.39, p=0.024; ‘treatment’: F(1,20)=6.30, p=0.033;‘genotype×treatment’: F(1,20)=1.81, p=0.21. **: p<0.01 TS vs. CO; #:p<0.05, ##: p<0.01 vehicle vs. 8581-treated mice; Bonferroni tests aftersignificant ANOVAs.

FIG. 14. 8581 rescued granular cell density in the hippocampus of TSmice. Data are expressed as means±S.E.M. of the density of DAPI+ cellsin the granular cell layer of vehicle- and 8581-treated TS and CO mice(A). ANOVA ‘genotype’: F(1, 20)=0.51, p=0.49; ‘treatment’: F(1,20)=7.09,p=0.026; ‘genotype×treatment’: F(1,20)=4.00, p=0.076. *: p<0.05 TS vs.CO; ##: p<0.01 vehicle vs. 8581-treated mice; Bonferroni tests aftersignificant ANOVAs. (B) Representative images of DAPI immunostaining ofvehicle- and 8581-treated TS and CO mice.

FIG. 15. 8581 normalized the percentage of area occupied by GAD+ buttonsin the hippocampus of TS mice. Data are expressed as means±S.E.M. of thepercentage of area occupied by GAD+ buttons in the hippocampus ofvehicle- and 8581-treated TS and CO mice (A). ANOVA ‘genotype’: F(1,20)=0.085, p=0.77; ‘treatment’: F(1,20)=1.14, p=0.30;‘genotype×treatment’: F(1,20)=7.15, p=0.017. *: p<0.05 TS vs. CO; #:p<0.05 vehicle vs. 8581-treated mice; Bonferroni tests after significantANOVAs. (B) Representative images of GAD immunostaining of vehicle- andR04938581-treated TS and CO mice.

FIG. 16. 8581 (1 mg/kg) rescues spatial learning deficits of Nf1+/−mice. Mean percentage of time spent in each quadrant during a probetrial (QL: left to target quadrant; QT: target quadrant; QR: right totarget quadrant; QO: opposite to target quadrant).

FIG. 17. 8581 (1 mg/kg) rescues spatial learning deficits of Nf1+/−mice. Average proximity to the target platform during a probe trial.

FIG. 18. 8581 (1 mg/kg) does not affect the performance of Nf1+/− miceunder conditions that occlude their behavioral deficits. Mean percentageof time spent in each quadrant during a probe trial (QL: left to targetquadrant; QT: target quadrant; QR: right to target quadrant; QO:opposite to target quadrant).

FIG. 19. 8581 (1 mg/kg) does not affect the performance of Nf1+/− miceunder conditions that occlude their behavioral deficits. Averageproximity to the target platform during a probe trial.

FIG. 20. Contextual conditioning: Dose response curve (0.3, 1.0 and 3.0mg/kg) of control mice treated with 8581 (P<0.05).

FIG. 21. Contextual conditioning of control mice when given 8581 (1mg/kg) for two consecutive days.

FIG. 22. Performance of control mice treated with vehicle or 8581 (1mg/kg) in the Rotarod.

FIG. 23. Performance of Nf1+/− mice treated with vehicle or 8581 (1mg/kg) in the Rotarod.

ABBREVIATIONS

ANOVA=analysis of varianceBZD=benzodiazepineCNS=central nervous systemCO=control

DS=Down Syndrome

F=F-test valueGABA=gamma-aminobutyric acidi.p.=intraperitonealLTP=long-term potentiationMANOVA=multivariate analysis of varianceMWM=Morris Water mazep=probabilityp.o.=peroralS.E.M.=standard error of the mean

TS=Ts65Dn

veh=vehicle

BRIEF DESCRIPTION OF THE TABLES

Table 1. Experimental groups of animals used in this invention forexamples 1 to 6.

Table 2a. Binding affinities and binding selectivities of activepharmaceutical compounds used in this invention.

Table 2b. Modulation of GABA A receptor subtypes expressed in Xenopusoocytes by active pharmaceutical compounds. Effect at human GABA A α5receptors: % change of a submaximal (EC10) response to GABA determinedat 30×Ki value from the flumazenil binding assay. Effect at human GABA Aα1, α2 and α3 receptors: % change of a submaximal (EC10) response toGABA determine at 3 μM or at 30×Ki value from the flumazenil bindingassay, if Ki was below 0.1 μM.

Table 3. Sensorimotor Test Battery (Mean Scores ±S.E.M.) of 8581-, R1-and vehicle-treated Ts65Dn and control mice.

Table 4. Hole Board test results (Mean Scores ±S.E.M.) of R1-, 8581- andvehicle-treated Ts65Dn and control mice. **: p<0.01 Ts65Dn vs. Control

Table 5. 8581 concentration in serum (ng/ml) of Nf1+/− and control mice0.5, 3, 7, and 24 hours after i.p. injection

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below.

The nomenclature used in this Application is based on IUPAC systematicnomenclature, unless indicated otherwise.

Any open valency appearing on a carbon, oxygen, sulfur or nitrogen atomin the structures herein indicates the presence of a hydrogen, unlessindicated otherwise.

The definitions described herein apply irrespective of whether the termsin question appear alone or in combination. It is contemplated that thedefinitions described herein can be appended to form chemically-relevantcombinations, such as e.g. “heterocycloalkyl-aryl”,“haloalkyl-heteroaryl”, “aryl-alkyl-heterocycloalkyl”, or“alkoxy-alkyl.” The last member of the combination is a radical which issubstituted by the other members of the combination in inverse order.

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogens by substituents.

The term “optional” or “optionally” denotes that a subsequentlydescribed event or circumstance may, but need not, occur and that thedescription includes instances where the event or circumstance occursand instances in which it does not.

The term “substituent” denotes an atom or a group of atoms replacing ahydrogen atom on the parent molecule.

The term “substituted” denotes that a specified group bears one or moresubstituents. Where any group can carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The term “compound(s) used in this invention” and “compound(s) usedpresent invention” refers to compounds of formula (I) or (II) andstereoisomers, tautomers, solvates, and salts (e.g., pharmaceuticallyacceptable salts) thereof.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, and polyamine resins.

The terms “halo,” “halogen,” and “halide” are used interchangeablyherein and denote fluoro, chloro, bromo, or iodo. Particularly, halorefers to F, Cl or Br, most particularly to F.

The term “alkyl” denotes a monovalent linear or branched saturatedhydrocarbon group of 1 to 12 carbon atoms, in particular of 1 to 7carbon atoms, more particular of 1 to 4 carbon atoms, for example,methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, ortert-butyl. Particularly, alkyl refers to methyl or isopropyl, mostparticularly to methyl.

The term “alkoxy” denotes a group of the formula —O—R′, wherein R′ is analkyl group. Examples of alkoxy moieties include methoxy, ethoxy,isopropoxy, and tert-butoxy.

The term “haloalkyl” denotes an alkyl group wherein at least one of thehydrogen atoms of the alkyl group has been replaced by same or differenthalogen atoms, particularly fluoro atoms. Examples of haloalkyl includemonofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, forexample 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl,fluoromethyl, or trifluoromethyl. The term “perhaloalkyl” denotes analkyl group where all hydrogen atoms of the alkyl group have beenreplaced by the same or different halogen atoms. Particularly, haloalkylrefers to monofluoromethyl and difluoromethyl.

The term “hydroxyalkyl” denotes an alkyl group wherein at least one ofthe hydrogen atoms of the alkyl group has been replaced by a hydroxygroup. Examples of hydroxyalkyl include hydroxymethyl, 2-hydroxyethyl,2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl,2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl,2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutylor 2-(hydroxymethyl)-3-hydroxypropyl.

The term “heterocycloalkyl” denotes a monovalent saturated or partlyunsaturated mono- or bicyclic ring system of 4 to 9 ring atoms,comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, theremaining ring atoms being carbon. Bicyclic means consisting of tworings having two ring atoms in common, i.e. the bridge separating thetwo rings is either a single bond or a chain of one or two ring atoms.Examples for monocyclic saturated heterocycloalkyl are azetidinyl,pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl,imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl,piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl,morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl,diazepanyl, homopiperazinyl, or oxazepanyl. Examples for bicyclicsaturated heterocycloalkyl are 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl,8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl,3-oxa-9-aza-bicyclo[3.3.1]nonyl, or 3-thia-9-aza-bicyclo[3.3.1]nonyl.Examples for partly unsaturated heterocycloalkyl are dihydrofuryl,imidazolinyl, dihydro-oxazolyl, tetrahydro-pyridinyl, or dihydropyranyl.Heterocycloalkyl can optionally be substituted as described herein.Particularly, heterocycloalkyl refers to morpholinyl, thiomorpholinyl,dioxothiomorpholinyl, 2-oxa-6-aza-spiro[3.3]hept-6-yl, pyrrolidinyl, andoxopyrrolidinyl. Most particularly, heterocycloalkyl refers tomorpholinyl, thiomorpholinyl, or dioxothiomorpholinyl.

The term “heterocycloalkylalkyl” denotes an alkyl group wherein at leastone of the hydrogen atoms of the alkyl group is replaced by aheterocycloalkyl group. Examples of heterocycloalkylalkyl includepyrrolidinyl-methyl, and pyrrolidinyl-methyl.

The term “aryl” denotes a monovalent aromatic carbocyclic mono- orbicyclic ring system comprising 6 to 10 carbon ring atoms. Examples ofaryl moieties include phenyl and naphthyl. Aryl can optionally besubstituted as described herein. Particular aryl is phenyl, andmonofluoro-phenyl.

The term “heteroaryl” denotes a monovalent aromatic heterocyclic mono-or bicyclic ring system of 5 to 12 ring atoms, comprising 1, 2, 3 or 4heteroatoms selected from N, O and S, the remaining ring atoms beingcarbon. Examples of heteroaryl moieties include pyrrolyl, furanyl,thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl,thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl,pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, benzofuranyl,isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranyl,benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl,benzoisothiazolyl, benzooxadiazolyl, benzothiadiazolyl, benzotriazolyl,purinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl,carbazolyl, or acridinyl. Heteroaryl can optionally be substituted asdescribed herein. Particularly, heteroaryl refers to pyridinyl,monofluoropyridinyl, and5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl, most particularly topyridinyl, and monofluoropyridinyl.

The term “oxo” denotes a divalent oxygen atom ═O.

The term “active pharmaceutical ingredient” (or “API”) denotes thecompound in a pharmaceutical composition that has a particularbiological activity.

The term “pharmaceutically acceptable” denotes an attribute of amaterial which is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic, and neither biologically nor otherwiseundesirable and is acceptable for veterinary as well as humanpharmaceutical use.

The term “pharmaceutically acceptable excipient” denotes any ingredienthaving no therapeutic activity and being non-toxic such asdisintegrators, binders, fillers, solvents, buffers, tonicity agents,stabilizers, antioxidants, surfactants or lubricants used in formulatingpharmaceutical products.

The term “pharmaceutical composition” (or “composition”) denotes amixture or solution comprising a therapeutically effective amount of anactive pharmaceutical ingredient together with pharmaceuticallyacceptable excipients to be administered to a mammal, e.g., a human inneed thereof.

The term “modulator” denotes a molecule that interacts with a targetreceptor. The interactions include e.g. agonistic, antagonistic, orinverse agonistic activity.

The term “inhibitor” denotes a compound which competes with, reduces orprevents the binding of a particular ligand to particular receptor orwhich reduces or prevents the inhibition of the function of a particularprotein.

The term “agonist” denotes a compound that has affinity to a receptorbinding site and which enhances the activity of the receptor-mediatedresponse as defined e.g. in Goodman and Gilman's “The PharmacologicalBasis of Therapeutics, 7th ed” in page 35, Macmillan Publ. Company,Canada, 1985. A “full agonist” effects a full response whereas a“partial agonist” effects less than full activation even when occupyingthe total receptor population. An “inverse agonist” produces an effectopposite to that of an agonist by binding to the same agonist bindingsite, or reduces the effect of an agonist by binding at a differentallosteric binding site.

The term “antagonist” denotes a compound that diminishes or prevents theaction of another compound or receptor site as defined e.g. in Goodmanand Gilman's “The Pharmacological Basis of Therapeutics, 7th ed.” inpage 35, Macmillan Publ. Company, Canada, 1985. In particular,antagonists refers to a compound that attenuates the effect of anagonist. A “competitive antagonist” binds to the same site as theagonist but does not activate it, thus blocks the agonist's action. A“non-competitive antagonist” binds to an allosteric (non-agonist) siteon the receptor to prevent activation of the receptor. A “reversibleantagonist” binds non-covalently to the receptor, therefore can be“washed out.” An “irreversible antagonist” binds covalently to thereceptor and cannot be displaced by either competing ligands or washing.

The term “allosteric modulator” denotes a compound that binds to areceptor at a site distinct from the agonist binding site (an“allosteric site”). It induces a conformational change in the receptor,which alters the affinity of the receptor for the endogenous ligand oragonist. “Positive allosteric modulators” increase the affinity, whilst“negative allosteric modulators” (NAM) decrease the affinity and hencedecrease the activity of a receptor indirectly. In present invention, anegative allosteric modulator particularly binds to the benzodiazepinebinding site with inverse agonism selective for GABA A α5subunit-containing receptor.

The term “inhibition constant” (Ki) denotes the absolute bindingaffinity of a particular inhibitor to a receptor. It is measured usingcompetition binding assays and is equal to the concentration where theparticular inhibitor would occupy 50% of the receptors if no competingligand (e.g. a radioligand) was present. Ki values can be convertedlogarithmically to pKi values (−log Ki), in which higher values indicateexponentially greater potency.

The term “submaximal effective concentration” (EC10) denotes theconcentration of a particular compound required for obtaining 10% of themaximum of a particular effect.

The term “binding selectivity” denotes the ratio between the bindingaffinity of a particular compound to two or more different receptorsubtypes. A particular compound is characterized as “binding selective”if its binding selectivity is 10 or more, more particularly if itsbinding selectivity is 20 or more, most particularly if its bindingselectivity is 50 or more.

The term “functional selectivity” denotes the different degrees ofmodulation by a particular compound at different receptor subtypes, e.g.by acting as an inverse agonist at one particular receptor subtypewhereas acting as an antagonist at another receptor subtype. In presentinvention, a compound is particularly functional selective if it acts asinverse agonist at GABA A α5β3γ2 receptor subtype by reducing the effectof GABA by more than 30% while affecting the other GABA A receptorsubtypes by less than 15%, particularly by less than 10%.

The terms “condition”, “defect”, “disability”, “disorder”, “disease” or“disease state” are used interchangeably to denote any disease,condition, symptom, disorder or indication.

The term “neurodevelopmental defect” denotes a disorder of neuraldevelopment, wherein growth and development of the brain or centralnervous system has been impaired (Reynolds C R et al., Handbook ofneurodevelopmental and genetic disorders in children (1999) GuilfordPress, N.Y.).

The term “GABAergic inhibition” refers to GABA mediatedneurotransmission which is inhibitory to mature neurons in vertebrates(Bernard C et al., Epilepsia (2000) 41(S6):S90-S95).

The term “excessive GABAergic inhibition” refers to increasedGABA-mediated neurotransmission which results in disruption ofexcitatory/inhibitory (E/I) circuit balance in favor of inhibition(Kleschevnikov A. M. et al., J. Neurosci. (2004) 24:8153-8160).

The term “cognitive deficit” or “cognitive impairment” describes anycharacteristic that acts as a barrier to cognitive performance. The termdenotes deficits in global intellectual performance, such as mentalretardation, it denotes specific deficits in cognitive abilities(learning disorders, dyslexia), or it denotes drug-induced memoryimpairment. Cognitive deficits can be congenital or caused byenvironmental factors such as brain injuries, neurological disorders, ormental illness. The term “cognitive deficit in Down Syndrome” or“cognitive impairment in Down Syndrome” denotes cognitive deficits insubjects exhibiting a triplication of chromosome 21, in particularabnormalities in learning, memory, and language that lead to mild toprofound impairment in intellectual functioning in such subjects.

The term “intellectual disability” (ID) or “mental retardation” denotesan early onset cognitive impairment expressed by a significantly reducedability to understand new or complex information, to learn new skills,with a reduced ability to cope independently, which started beforeadulthood, with a lasting effect on development.

The term “procognitive” describes any characteristic that reduces orreverts conditions such as mental confusion, disorientation, delirium orcognitive deficits or that improves cognition.

The term “neurofibromatosis type 1” (NF1) denotes a disorder that iscaused by a mutation of a gene on chromosome 17 which encodes a proteinknown as neurofibromin relevant in intracellular signaling (Cui Y etal., Cell (2008) 135: 549-60).

The term “autism” denotes a disorder of neural development characterizedby impaired social interaction and communication, and by restricted andrepetitive behavior (American Psychiatric Association Inc., Diagnosticand Statistical Manual of Mental Disorders (DSM-IV-TR) (2000) 4th ed.).

The term “stroke” is the rapidly developing loss of brain function(s)due to disturbance in the blood supply to the brain. This can be due toischemia (lack of blood flow) caused by blockage (thrombosis, arterialembolism), or a hemorrhage (Sims N R et al, Biochimica et BiophysicaActa (2009) 1802(1):80-91).

The term “recovery after stroke” refers to the ability to restoreimpaired brain function after stroke (Dimyan M.A. et al., Nat RevNeurol. (2011) 7(2): 76-85).

The term “treating” or “treatment” of a disease state includes (1)preventing the disease state, i.e. causing the clinical symptoms of thedisease state not to develop in a subject that may be exposed to orpredisposed to the disease state, but does not yet experience or displaysymptoms of the disease state, (2) inhibiting the disease state, i.e.,arresting the development of the disease state or its clinical symptoms,or (3) relieving the disease state, i.e., causing temporary or permanentregression of the disease state or its clinical symptoms.

The term “therapeutically effective amount” denotes an amount of acompound of the present invention that, when administered to a subject,(i) treats or prevents the particular disease, condition or disorder,(ii) attenuates, ameliorates or eliminates one or more symptoms of theparticular disease, condition, or disorder, or (iii) prevents or delaysthe onset of one or more symptoms of the particular disease, conditionor disorder described herein. The therapeutically effective amount willvary depending on the compound, disease state being treated, theseverity or the disease treated, the age and relative health of thesubject, the route and form of administration, the judgement of theattending medical or veterinary practitioner, and other factors.

The term “subject” or “patient” denotes an animal, more particularly avertebrate. In certain embodiments, the vertebrate is a mammal. Mammalsinclude humans, non-human primates such as chimpanzees and other apesand monkey species, farm animals such as cattle, horses, sheep, goats,and swine, domestic animals such as rabbits, dogs, and cats, laboratoryanimals including rodents, such as rats, mice, and guinea pigs. Incertain embodiments, a mammal is a human. The term subject does notdenote a particular age or sex.

In detail, the present invention provides the use of a GABA A α5negative allosteric modulator for the treatment, prevention and/or delayof progression of central nervous system (CNS) conditions caused byneurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions causedby neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits in Down Syndrome, in autism, inneurofibromatosis type I, or after stroke.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions causedby neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits in Down Syndrome.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions causedby neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits in autism.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions causedby neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits in neurofibromatosis type I.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions causedby neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits after stroke.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions causedby neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from intellectual disability.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is a ligand to a BZD binding site and is acting asinverse agonist at GABA A α5 subunit-containing receptors.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is a ligand to the BZD binding site of the GABA Areceptor and is acting as inverse agonist at the GABA A α5β3γ2 receptorsubtype.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator has binding selectivity at GABA A α5subunit-containing receptors.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator has functional selectivity at GABA A α5subunit-containing receptors.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is binding to human GABA A α5β3γ2 receptor subtypewith a binding selectivity of a factor of 10 or more as compared tobinding affinities to human GABA A α1β2/3γ2, α2β3γ2 and α3β3γ2 receptorsubtypes.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator has binding selectivity at GABA A α5subunit-containing receptors.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator exhibits a functional selectivity by acting asinverse agonist at human GABA A α5β3γ2 receptor subtype by reducing theeffect of GABA by more than 30% and in addition affecting the effect ofGABA at human GABA A α1β2/3γ2, α2β3γ2 and α3β3γ2 receptor subtypes byless than 15%.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is selected from a compound of formula (I) or acompound of formula (II)

whereinR¹ is hydrogen, halo, alkyl, haloalkyl, or cyano;R² is hydrogen, halo, alkyl, haloalkyl, or cyano;R³ is hydrogen, alkyl, or heterocycloalkylalkyl, whereinheterocycloalkylalkyl is optionally substituted with one or morehydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, or cyano;R⁴ is aryl or heteroaryl, each optionally substituted by one, two orthree halo;R⁵ is hydrogen, alkyl, haloalkyl or hydroxyalkyl;R⁶ is —C(O)—NR⁷R⁸R⁷ is hydrogen;R⁸ is alkyl;or R⁷ and R⁸ together with the nitrogen to which they are bound form aheterocycloalkyl, or a heteroaryl, each optionally substituted with oneor more hydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, orcyano;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is selected from a compound of formula (I) wherein

R¹ is hydrogen, halo, alkyl, haloalkyl, or cyano;R² is hydrogen, halo, alkyl, haloalkyl, or cyano;R³ is hydrogen, alkyl, or heterocycloalkylalkyl, whereinheterocycloalkylalkyl is optionally substituted with one or morehydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, or cyano;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is selected from a compound of formula (I) wherein

R¹ is hydrogen, halo, haloalkyl, or cyano;R² is halo, or haloalkyl;R³ is hydrogen, alkyl, or heterocycloalkylalkyl substituted with oneoxo;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is selected from:

-   3-Fluoro-10-fluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Cyano-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Chloro-10-fluoromethyl-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3,10-Dichloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-3-cyano-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-3-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-chloro-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Bromo-3-fluoro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-methyl-6-(2-oxo-pyrrolidin-1-ylmethyl)-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;    or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is not3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is selected from a compound of formula (II) wherein

R⁴ is aryl or heteroaryl, each optionally substituted by one, two orthree halo;R⁵ is hydrogen, alkyl, haloalkyl or hydroxyalkyl;R⁶ is —C(O)—NR⁷R⁸;R⁷ is hydrogen;R⁸ is alkyl;or R⁷ and R⁸ together with the nitrogen to which they are bound form aheterocycloalkyl, or a heteroaryl, each optionally substituted with oneor more hydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, orcyano;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is selected from a compound of formula (II) wherein

R⁴ is aryl or heteroaryl, each optionally substituted by one halo;R⁵ is alkyl;R⁶ is C(O)—NR⁷R⁸;R⁷ is hydrogen and R⁸ is alkyl;or R⁷ and R⁸ together with the nitrogen to which they are bound form aheterocycloalkyl optionally substituted with one or two oxo, or form aheteroaryl;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is selected from:N-Isopropyl-6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-nicotinamide;

-   (5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-[6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-methanone;-   [6-(5-Methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-methanone;-   (1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;-   {6-[3-(4-Chloro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-morpholin-4-yl-methanone;-   [6-(5-Methyl-3-pyridin-2-yl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-morpholin-4-yl-methanone;-   6-[3-(5-Fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-isopropyl-nicotinamide;-   (1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(5-fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;-   {6-[3-(5-Chloro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-thiomorpholin-4-yl-methanone;    or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator isN-Isopropyl-6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-nicotinamide; ora pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is(5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-[6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is[6-(5-Methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is{6-[3-(4-Chloro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-morpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is[6-(5-Methyl-3-pyridin-2-yl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-morpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is6-[3-(5-Fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-isopropyl-nicotinamide;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(5-fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is{6-[3-(5-Chloro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-thiomorpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator, wherein the GABA A α5 negativeallosteric modulator is used separately, sequentially or simultaneouslyin combination with a second active pharmaceutical compound.

In a particular embodiment, the invention provides a method for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions caused by neurodevelopmental defects whichresult in excessive GABAergic inhibition in the cortex and hippocampusin a subject in need of such treatment, which comprises administering tosaid subject a therapeutically effective amount of a GABA A α5 negativeallosteric modulator as described herein in a pharmaceuticallyacceptable form.

In a particular embodiment, the invention provides a method for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromcognitive deficits in Down Syndrome, in a subject in need of suchtreatment, which comprises administering to said subject atherapeutically effective amount of a GABA A α5 negative allostericmodulator as described herein in a pharmaceutically acceptable form.

In a particular embodiment, the invention provides a method for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromcognitive deficits in autism, in a subject in need of such treatment,which comprises administering to said subject a therapeuticallyeffective amount of a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form.

In a particular embodiment, the invention provides a method for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromcognitive deficits in neurofibromatosis type I, in a subject in need ofsuch treatment, which comprises administering to said subject atherapeutically effective amount of a GABA A α5 negative allostericmodulator as described herein in a pharmaceutically acceptable form.

In a particular embodiment, the invention provides a method for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromcognitive deficits after stroke, in a subject in need of such treatment,which comprises administering to said subject a therapeuticallyeffective amount of a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form.

In a particular embodiment, the invention provides a method for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromintellectual disability, in a subject in need of such treatment, whichcomprises administering to said subject a therapeutically effectiveamount of a GABA A α5 negative allosteric modulator as described hereinin a pharmaceutically acceptable form.

In a particular embodiment, the invention provides a pharmaceuticalcomposition comprising a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions caused by neurodevelopmental defects whichresult in excessive GABAergic inhibition in the cortex and hippocampus.

In a particular embodiment, the invention provides a pharmaceuticalcomposition comprising a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromcognitive deficits in Down Syndrome.

In a particular embodiment, the invention provides a pharmaceuticalcomposition comprising a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromcognitive deficits in autism.

In a particular embodiment, the invention provides a pharmaceuticalcomposition comprising a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromcognitive deficits in neurofibromatosis type I.

In a particular embodiment, the invention provides a pharmaceuticalcomposition comprising a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromcognitive deficits after stroke.

In a particular embodiment, the invention provides a pharmaceuticalcomposition comprising a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form for thetreatment, prevention and/or delay of progression of central nervoussystem (CNS) conditions, wherein said CNS condition is selected fromintellectual disability.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described herein for the preparationof medicaments for the treatment, prevention and/or delay of progressionof central nervous system (CNS) conditions caused by neurodevelopmentaldefects which result in excessive GABAergic inhibition in the cortex andhippocampus.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described herein for the preparationof medicaments for the treatment, prevention and/or delay of progressionof central nervous system (CNS) conditions, wherein said CNS conditionis selected from cognitive deficits in Down Syndrome.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described herein for the preparationof medicaments for the treatment, prevention and/or delay of progressionof central nervous system (CNS) conditions, wherein said CNS conditionis selected from cognitive deficits in autism.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described herein for the preparationof medicaments for the treatment, prevention and/or delay of progressionof central nervous system (CNS) conditions, wherein said CNS conditionis selected from cognitive deficits in neurofibromatosis type I.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described herein for the preparationof medicaments for the treatment, prevention and/or delay of progressionof central nervous system (CNS) conditions, wherein said CNS conditionis selected from cognitive deficits after stroke.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described herein for the preparationof medicaments for the treatment, prevention and/or delay of progressionof central nervous system (CNS) conditions, wherein said CNS conditionis selected from intellectual disability.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionscaused by neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditions,wherein said CNS condition is selected from cognitive deficits in DownSyndrome.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditions,wherein said CNS condition is selected from cognitive deficits inautism.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditions,wherein said CNS condition is selected from cognitive deficits inneurofibromatosis type I.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditions,wherein said CNS condition is selected from cognitive deficits afterstroke

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditions,wherein said CNS condition is selected from intellectual disability. Ina particular embodiment, the invention provides the use of a GABA A α5negative allosteric modulator for the treatment, prevention and/or delayof progression of central nervous system (CNS) conditions caused byneurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits in Down Syndrome, and wherein the GABAA α5 negative allosteric modulator is3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine,or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions causedby neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits in Down Syndrome, and wherein the GABAA α5 negative allosteric modulator is(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator for the treatment, prevention and/or delay ofprogression of central nervous system (CNS) conditions caused byneurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits in Down Syndrome, and wherein the GABAA α5 negative allosteric modulator is3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine,or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the a GABA A α5negative allosteric modulator for the treatment, prevention and/or delayof progression of central nervous system (CNS) conditions caused byneurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus, wherein said CNS condition isselected from cognitive deficits in Down Syndrome, and wherein the GABAA α5 negative allosteric modulator is(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides the use of a GABA A α5negative allosteric modulator selected from a compound of formula (I) ora compound of formula (II)

whereinR¹ is hydrogen, halo, alkyl, haloalkyl, or cyano;R² is hydrogen, halo, alkyl, haloalkyl, or cyano;R³ is hydrogen, alkyl, or heterocycloalkylalkyl, whereinheterocycloalkylalkyl is optionally substituted with one or morehydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, or cyano;R⁴ is aryl or heteroaryl, each optionally substituted by one, two orthree halo;R⁵ is hydrogen, alkyl, haloalkyl or hydroxyalkyl;R⁶ is —C(O)—NR⁷R⁸R⁷ is hydrogen;R⁸ is alkyl;or R⁷ and R⁸ together with the nitrogen to which they are bound form aheterocycloalkyl, or a heteroaryl, each optionally substituted with oneor more hydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, orcyano;or a pharmaceutically acceptable salt thereof;for the treatment, prevention and/or delay of progression of centralnervous system (CNS) conditions related to excessive GABAergicinhibition in the cortex and hippocampus.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described above for the treatment,prevention and/or delay of progression of central nervous system (CNS)conditions related to excessive GABAergic inhibition in the cortex andhippocampus, wherein the excessive GABAergic inhibition in the thecortex and hippocampus is caused by neurodevelopmental defects.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described above for the treatment,prevention and/or delay of progression of central nervous system (CNS)conditions caused by neurodevelopmental defects which result inexcessive GABAergic inhibition in the cortex and hippocampus.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described above for the treatment,prevention and/or delay of progression of central nervous system (CNS)conditions related to excessive GABAergic inhibition in the cortex andhippocampus, particularly caused by neurodevelopmental defects, whereinsaid CNS condition is selected from cognitive deficits in Down Syndrome,in autism or in neurofibromatosis type I.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described above for the treatment,prevention and/or delay of progression of central nervous system (CNS)conditions related to excessive GABAergic inhibition in the cortex andhippocampus, particularly caused by neurodevelopmental defects, whereinsaid CNS condition is cognitive deficits in Down Syndrome.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described above for the treatment,prevention and/or delay of progression of central nervous system (CNS)conditions related to excessive GABAergic inhibition in the cortex andhippocampus, particularly caused by neurodevelopmental defects, whereinsaid CNS condition is cognitive deficits in autism.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described above for the treatment,prevention and/or delay of progression of central nervous system (CNS)conditions related to excessive GABAergic inhibition in the cortex andhippocampus, particularly caused by neurodevelopmental defects, whereinsaid CNS condition is cognitive deficits in neurofibromatosis type I.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described above for the treatment,prevention and/or delay of progression of central nervous system (CNS)conditions related to excessive GABAergic inhibition in the cortex andhippocampus, wherein said CNS condition is characterized by disabilitiesafter stroke.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris binding to human GABA A α5β3γ2 receptor subtype with a bindingselectivity of a factor of 10 or more as compared to binding affinitiesto human GABA A α1β2/3γ2, α2β3γ2 and α3β3γ2 receptor subtypes.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatorexhibits a functional selectivity by acting as inverse agonist at humanGABA A α5β3γ2 receptor subtype by reducing the effect of GABA by morethan 30% and in addition affecting the effect of GABA at human GABA Aα1β2/3γ2, α2β3γ2 and α3β3γ2 receptor subtypes by less than 15%.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris selected from a compound of formula (I), wherein R¹, R², and R³ areas defined herein, or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris selected from a compound of formula (I), wherein R¹ is hydrogen,halo, haloalkyl, or cyano; R² is halo, or haloalkyl; R³ is hydrogen,alkyl, or heterocycloalkylalkyl substituted with one oxo; or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris selected from

-   3-Fluoro-10-fluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Cyano-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Chloro-10-fluoromethyl-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3,10-Dichloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-3-cyano-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-3-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-chloro-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Bromo-3-fluoro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-methyl-6-(2-oxo-pyrrolidin-1-ylmethyl)-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;    or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris selected from a compound of formula (II) wherein R⁴, R⁵, and R⁶ areas defined herein, or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris selected from a compound of formula (II) wherein R⁴ is aryl orheteroaryl, each optionally substituted by one halo; R⁵ is alkyl; R⁶ isC(O)—NR⁷R⁸; R⁷ is hydrogen and R⁸ is alkyl; or R⁷ and R⁸ together withthe nitrogen to which they are bound form a heterocycloalkyl optionallysubstituted with one or two oxo, or form a heteroaryl; or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris selected from:

-   N-Isopropyl-6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-nicotinamide;-   (5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-[6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-methanone;-   [6-(5-Methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-methanone;-   (1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;-   {6-[3-(4-Chloro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-morpholin-4-yl-methanone;-   [6-(5-Methyl-3-pyridin-2-yl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-morpholin-4-yl-methanone;-   6-[3-(5-Fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-isopropyl-nicotinamide;-   (1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(5-fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;-   {6-[3-(5-Chloro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-thiomorpholin-4-yl-methanone;    or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris N-Isopropyl-6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-nicotinamide;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris(5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-[6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris[6-(5-Methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris{6-[3-(4-Chloro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-morpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris[6-(5-Methyl-3-pyridin-2-yl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-morpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris6-[3-(5-Fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-isopropyl-nicotinamide;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(5-fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris{6-[3-(5-Chloro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-thiomorpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator for the treatment, prevention and/ordelay of progression of central nervous system (CNS) conditions asdescribed herein, wherein said GABA A α5 negative allosteric modulatoris used separately, sequentially or simultaneously in combination with asecond active pharmaceutical compound.

In a particular embodiment, the invention provides a method for thetreatment, prevention and/or delay of progression of cognitive deficitsin Down Syndrome, in autism, in neurofibromatosis type I, or forrecovery after stroke in a subject in need of such treatment, whichcomprises administering to said subject a therapeutically effectiveamount of a GABA A α5 negative allosteric modulator as described hereinin a pharmaceutically acceptable form.

In a particular embodiment, the invention provides a pharmaceuticalcomposition comprising a GABA A α5 negative allosteric modulator asdescribed herein in a pharmaceutically acceptable form for thetreatment, prevention and/or delay of progression of cognitive deficitsin Down Syndrome, in autism, in neurofibromatosis type I, or forrecovery after stroke.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of cognitive deficits in Down Syndrome, inautism, in neurofibromatosis type I, or for recovery after stroke.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the preparation ofmedicaments for the treatment, prevention and/or delay of progression ofcognitive deficits in Down Syndrome, in autism, in neurofibromatosistype I, or for recovery after stroke.

In a particular embodiment, the invention provides the use of a GABA Aα5 negative allosteric modulator as described herein for the preparationof medicaments for the treatment, prevention and/or delay of progressionof cognitive deficits in Down Syndrome, in autism, in neurofibromatosistype I, or for recovery after stroke.

In another embodiment, the invention provides a GABA A α5 negativeallosteric modulator selected from a compound of formula (I) or acompound of formula (II)

whereinR¹ is hydrogen, halo, alkyl, haloalkyl, or cyano;R² is hydrogen, halo, alkyl, haloalkyl, or cyano;R³ is hydrogen, alkyl, or heterocycloalkylalkyl, whereinheterocycloalkylalkyl is optionally substituted with one or morehydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, or cyano;R⁴ is aryl or heteroaryl, each optionally substituted by one, two orthree halo;R⁵ is hydrogen, alkyl, haloalkyl or hydroxyalkyl;R⁶ is —C(O)—NR⁷R⁸R⁷ is hydrogen;R⁸ is alkyl;or R⁷ and R⁸ together with the nitrogen to which they are bound form aheterocycloalkyl, or a heteroaryl, each optionally substituted with oneor more hydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, orcyano;or a pharmaceutically acceptable salt thereof;for the treatment, prevention and/or delay of progression of centralnervous system (CNS) conditions related to excessive GABAergicinhibition in the cortex and hippocampus

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsrelated to excessive GABAergic inhibition in the cortex and hippocampus,which is caused by neurodevelopmental defects.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionscaused by neurodevelopmental defects which result in excessive GABAergicinhibition in the cortex and hippocampus.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsrelated to excessive GABAergic inhibition in the cortex and hippocampus,particularly caused by neurodevelopmental defects, wherein said CNScondition is selected from cognitive deficits in Down Syndrome, inautism or in neurofibromatosis type I.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsrelated to excessive GABAergic inhibition in the cortex and hippocampus,particularly caused by neurodevelopmental defects, wherein said CNScondition is cognitive deficits in Down Syndrome.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsrelated to excessive GABAergic inhibition in the cortex and hippocampus,particularly caused by neurodevelopmental defects, wherein said CNScondition is cognitive deficits in autism.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsrelated to excessive GABAergic inhibition in the cortex and hippocampus,particularly caused by neurodevelopmental defects, wherein said CNScondition is cognitive deficits in neurofibromatosis type I.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsrelated to excessive GABAergic inhibition in the cortex and hippocampus,wherein said CNS condition is characterized by disabilities afterstroke.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is binding to human GABA A α5β3γ2 receptor subtype with abinding selectivity of a factor of 10 or more as compared to bindingaffinities to human GABA A α1β2/3γ2, α2β3γ2 and α3β3γ2 receptorsubtypes.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator exhibits a functional selectivity by acting as inverse agonistat human GABA A α5β3γ2 receptor subtype by reducing the effect of GABAby more than 30% and in addition affecting the effect of GABA at humanGABA A α1β2/3γ2, α2β3γ2 and α3β3γ2 receptor subtypes by less than 15%.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is selected from a compound of formula (I), wherein R¹, R²,and R³ are as defined herein, or a pharmaceutically acceptable saltthereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is selected from a compound of formula (I), wherein R¹ ishydrogen, halo, haloalkyl, or cyano; R² is halo, or haloalkyl; R³ ishydrogen, alkyl, or heterocycloalkylalkyl substituted with one oxo; or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is selected from

-   3-Fluoro-10-fluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Cyano-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Chloro-10-fluoromethyl-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3,10-Dichloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-3-cyano-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Chloro-3-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-chloro-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   10-Bromo-3-fluoro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;-   3-Bromo-10-methyl-6-(2-oxo-pyrrolidin-1-ylmethyl)-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;    or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepineor a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is not3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepineor a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is selected from a compound of formula (II) wherein R⁴, R⁵,and R⁶ are as defined herein, or a pharmaceutically acceptable saltthereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is selected from a compound of formula (II) wherein R⁴ is arylor heteroaryl, each optionally substituted by one halo; R⁵ is alkyl; R⁶is C(O)—NR⁷R⁸; R⁷ is hydrogen and R⁸ is alkyl; or R⁷ and R⁸ togetherwith the nitrogen to which they are bound form a heterocycloalkyloptionally substituted with one or two oxo, or form a heteroaryl; or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is selected from:

-   N-Isopropyl-6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-nicotinamide;-   (5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-[6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-methanone;-   [6-(5-Methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-methanone;-   (1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;-   {6-[3-(4-Chloro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-morpholin-4-yl-methanone;-   [6-(5-Methyl-3-pyridin-2-yl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-morpholin-4-yl-methanone;-   6-[3-(5-Fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-isopropyl-nicotinamide;-   (1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(5-fluoro-pyridin-2-yl-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;-   {6-[3-(5-Chloro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-thiomorpholin-4-yl-methanone;    or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulatorN-Isopropyl-6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-nicotinamide; ora pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is(5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-[6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is[6-(5-Methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanoneor a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is{6-[3-(4-Chloro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-morpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is[6-(5-Methyl-3-pyridin-2-yl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-morpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is6-[3-(5-Fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-isopropyl-nicotinamide;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(5-fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is{6-[3-(5-Chloro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-thiomorpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the invention provides a GABA A α5 negativeallosteric modulator as described herein for the treatment, preventionand/or delay of progression of central nervous system (CNS) conditionsas described herein, wherein said GABA A α5 negative allostericmodulator is used separately, sequentially or simultaneously incombination with a second active pharmaceutical compound.

EXAMPLES Materials and Methods

a. Animals

Table 1 shows the number of male animals that were used in this study.Ten control and ten Ts65Dn mice of 6 months of age at the beginning ofthe treatment received 8581; 16 control and 15 Ts65Dn mice of 5-6 monthsof age at the beginning of the treatment received R1, the other twogroups of control (n=13) and Ts65Dn (n=13) mice received vehicle.

b. Active Pharmaceutical Compounds

Active pharmaceutical compounds used in present invention were preparedas described previously in WO 2006/045429, WO 2006/045430, WO2007/042421 and WO 2009/071476:

Compound 8580

3-Fluoro-10-fluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045430 on page 17 in Example 3.

Compound 8581

3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045430 on page 21 in Example 7.

Compound 8582

3-Cyano-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045430 on page 23 in Example 13.

Compound 8583

10-Difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045430 on page 26 in Example 16.

Compound 8584

3-Chloro-10-fluoromethyl-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045430 on page 28 in Example 20.

Compound 8585

10-Chloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine wasprepared as described in WO 2006/045429 on page 15 in Example 1.

Compound 8586

3,10-Dichloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045429 on page 23 in Example 20.

Compound 8587

10-Chloro-3-cyano-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045429 on page 37 in Example 47.

Compound 8588

10-Chloro-3-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045429 on page 29 in Example 32.

Compound 8589

3-Bromo-10-chloro-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2006/045429 on page 33 in Example 38.

Compound 8590

10-Bromo-3-fluoro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine was prepared asdescribed in WO 2006/045429 on page 37 in Example 47.

Compound 8591

3-Bromo-10-methyl-6-(2-oxo-pyrrolidin-1-ylmethyl)-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepinewas prepared as described in WO 2007/042421 on page 67 in Example 101.

Compound O1

N-Isopropyl-6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-nicotinamide wasprepared as described in WO 2009/071476 on page 50 in Example 26.

Compound P1

(5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-[6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-methanonewas prepared as described in WO 2009/071476 on page 62 in Example 75.

Compound Q1

[6-(5-Methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-methanonewas prepared as described in WO 2009/071476 on page 64 in Example 81.

Compound R1

(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanonewas prepared as described in WO 2009/071476 on page 75 in Example 112.

Compound S1

{6-[3-(4-Chloro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-morpholin-4-yl-methanonewas prepared as described in WO 2009/071476 on page 78 in Example 123.

Compound T1

[6-(5-Methyl-3-pyridin-2-yl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-morpholin-4-yl-methanonewas prepared as described in WO 2009/071476 on page 123 in Example 274.

Compound U1

6-[3-(5-Fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-isopropyl-nicotinamidewas prepared as described in WO 2009/071476 on page 127 in Example 289.

Compound V1

(1,1-Dioxo-1λ6-thiomorpholin-4-yl)-{6-[3-(5-fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanonewas prepared as described in WO 2009/071476 on page 127 in Example 293.

Compound W1

{6-[3-(5-Chloro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-thiomorpholin-4-yl-methanonewas prepared as described in WO 2009/071476 on page 132 in Example 310.

The binding affinities of the above active pharmaceutical compounds atthe GABA A receptor subtypes have been measured by competition for[³H]flumazenil (85 Ci/mmol; Roche) binding to HEK293 cells expressingrat (stably transfected) or human (transiently transfected) receptors ofcomposition α1β2/3γ2, α2β3γ2, α3β3γ2, and α5β3γ2. As can be seen fromTable 2a, active pharmaceutical compounds used in this invention exhibithigh affinity at the α5β3γ2 receptor subtype and good selectivity overα1β2/3γ2, α2β3γ2 and α3β3γ2 receptor subtypes.

As can be seen from Table 2b, the active pharmaceutical compounds usedin present invention also demonstrate a substantial functionalselectivity. Subtype-selective effects were determined on clonedreceptors expressed in Xenopus oocytes. Human recombinant GABA Areceptors were expressed in Xenopus laevis oocytes. Current responseswere evoked in two-microelectrode voltage-clamp condition by applying anEC10 of GABA before and during the co-application of the test compound.Response amplitudes in the presence of the test compound are expressedas percentage of the amplitudes before drug addition.

c. Pharmaceutical Compositions

For mice studies, active pharmaceutical compounds of present inventionwere formulated in chocolate-milk (Puleva, Barcelona, Spain). Activepharmaceutical compounds of present invention or vehicle wereadministered p.o. at a dose of 20 mg/kg for six weeks. Theiradministration was prolonged during the 30 days of the behavioralassessment.

For human use pharmaceutical compositions or medicaments can be preparedcomprising active pharmaceutical compounds as described above and atherapeutically inert carrier, diluent or excipient, as well as methodsof using the compounds of the invention to prepare such compositions andmedicaments.

Compositions are formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners.

The active pharmaceutical compounds used in the invention can beadministered by any suitable means, including oral, topical (includingbuccal and sublingual), rectal, vaginal, transdermal, parenteral,subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecaland epidural and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration.

The active pharmaceutical compounds used in the invention can beadministered in any convenient administrative form, e.g., tablets,powders, capsules, solutions, dispersions, suspensions, syrups, sprays,suppositories, gels, emulsions, patches, etc. Such compositions cancomprise components conventional in pharmaceutical preparations, e.g.,diluents, carriers, pH modifiers, preservatives, solubilizers,stabilizers, wetting agents, emulsifiers, sweeteners, colorants,flavorants, salts for varying the osmotic pressure, buffers, maskingagents, antioxidants, and further active agents. They can also comprisestill other therapeutically valuable substances.

A typical pharmaceutical composition is prepared by mixing an activepharmaceutical compounds used in the invention t and a carrier orexcipient. Suitable carriers and excipients are well known to thoseskilled in the art and are described in detail in, e.g., Ansel H. C. etal., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems(2004) Lippincott, Williams & Wilkins, Philadelphia; Gennaro A. R. etal., Remington: The Science and Practice of Pharmacy (2000) Lippincott,Williams & Wilkins, Philadelphia; and Rowe R. C, Handbook ofPharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. Thepharmaceutical compositions can also include one or more buffers,stabilizing agents, surfactants, wetting agents, lubricating agents,emulsifiers, suspending agents, preservatives, antioxidants, opaquingagents, glidants, processing aids, colorants, sweeteners, perfumingagents, flavoring agents, diluents and other known additives to providean elegant presentation of the drug (i.e., a compound of the presentinvention or pharmaceutical composition thereof) or aid in themanufacturing of the pharmaceutical product (i.e., medicament).

The dosage at which active pharmaceutical compounds used in theinvention can be administered can vary within wide limits and will, ofcourse, be fitted to the individual requirements in each particularcase. In general, in the case of oral administration a daily dosage ofabout 0.1 to 1500 mg, more particular 1 to 1000 mg, most particular 5 to500 mg per person of an active pharmaceutical compounds used in theinvention should be appropriate, although the above upper limit can alsobe exceeded when necessary.

An example of a suitable oral dosage form is a tablet comprising about100 mg to 500 mg of an active pharmaceutical compounds used in theinvention compounded with about 90 to 30 mg anhydrous lactose, about 5to 40 mg sodium croscarmellose, about 5 to 30 mg polyvinylpyrrolidone(PVP) K30, and about 1 to 10 mg magnesium stearate. The powderedingredients are first mixed together and then mixed with a solution ofthe PVP. The resulting composition can be dried, granulated, mixed withthe magnesium stearate and compressed to tablet form using conventionalequipment.

An example of an aerosol composition can be prepared by dissolving anactive pharmaceutical compounds used in the invention, for example 10 to100 mg, in a suitable buffer solution, e.g. a phosphate buffer, adding atonicifier, e.g. a salt such as sodium chloride, if desired. Thesolution can be filtered, e.g., using a 0.2 μm filter, to removeimpurities and contaminants.

d. Statistical Analysis

The data were analyzed using two-way (‘genotype’×‘treatment’) ANOVAs.The Morris water maze data were analyzed using a two-way ANOVA withrepeated measures (‘session’×‘genotype’×‘treatment’). The means of eachexperimental group were compared post-hoc by Student's t-test if twogroups were compared or Bonferroni tests if more than two groups werecompared. All the analyses were done using SPSS for Windows version 17.0(SPSS AG, Zurich, Switzerland).

Example 1. Sensorimotor Tests

A battery of sensorimotor tests was performed following the proceduredescribed by Rueda N et al. [Neurosci Lett (2008) 433(1): 22-27]. In thevisual placing reflex test, cerebellar and vestibular functions wereevaluated. In 3 consecutive trials, mice were gently lowered by the tailtowards a flat surface from a height of 15 cm. The response of forepawextension was scored on a 0-4 scale [4: animal extends the forepaws whenplaced at the highest height; 3: forepaws extended before touching thesurface with vibrissae; 2: forepaws extended after vibrissae touched thesurface; 1: forepaws extended after the nose touched the surface; 0: noextension].

To evaluate auditory sensitivity, the startle response to a suddenauditory stimulus was measured. Mice were placed facing the wall of anunfamiliar cage and the auditory stimulus was generated by clappingtogether two stainless steel forceps (7 cm long). A score (0-3 points)was assigned based on the magnitude of the response: jumping more than 1cm (3 points); jumping less than 1 cm (2 points); retracting of the ears(Preyer reflex, 1 point); or no response (0 points).

The vibrissa placing reflex was analyzed by noting the reflectivereaction to touching the vibrissae with a cotton stick. In threeconsecutive trials, a score of 1 was assigned to animals that touchedthe stimulated vibrissae with an ipsilateral paw, and 0 if there was noresponse. Grip strength was assessed by quantifying the resistance tobeing separated from a lid of aluminum bars (2 mm), when dragged by thetail (0: no resistance, total loss of grip strength; 1: slight; 2:moderate; 3: active; 4: extremely active resistance, normal gripstrength).

In order to evaluate equilibrium, four 20-s trials of balance wereperformed on an elevated (40 cm high), horizontal (50 cm long) rod.Trials 1 and 2 were performed on a flat wooden rod (9 mm wide); trials 3and 4 were performed on a cylindrical aluminum rod (1 cm diameter). Ineach trial, the animals were placed in a marked central zone (10 cm) onthe elevated rod. A score of 0 was given if the animal fell within 20 s,1 if it stayed within the central zone for >20 s, 2 if it left thecentral zone, and 3 if it reached one of the ends of the bar.

Prehensile reflex (three 5-s trials) was measured as the ability of theanimal to remain suspended by the forepaws by grasping an elevatedhorizontal wire (2 mm in diameter). The maximum possible score of 3 wasachieved when the animal remained suspended by the forepaws in all threetrials (one point per trial). Traction capacity was scored at the sametime by assessing the number of hind limbs that the animal raised toreach the wire (0: none; 1: one limb; 2: two limbs).

Table 3 shows the score of 8581-, R1- and vehicle-treated Ts65Dn andcontrol mice in the different sensorimotor tests. 8581 or R1 treatmentdid not modify any of the sensorimotor abilities tested in Ts65Dn orcontrol mice (vision, audition, strength, equilibrium prehensile reflex,traction capacity or motor coordination in the coat hanging test).

Example 2. Motor Coordination: Rotarod

Motor coordination was evaluated using a rotarod device (Ugo Basile;Comerio, Italy), which consists of a 37-cm-long, 3-cm diameter plasticrod that rotates at different speeds. In a single session, 4 trials witha maximum duration of 60 s each were performed. In the first threetests, the rod was rotated at constant speeds of 5, 25 and 50 rpm,respectively. The last trial consisted of an acceleration cycle, inwhich the rod rotated progressively faster, and the animal had to adaptto the growing demands of the test. The length of time that each animalstayed on the rotarod was recorded.

As shown in FIGS. 1 and 2 motor coordination in the rotarod was notmodified in mice of either genotype after 8581- or R1-treatment. Ts65Dnand control mice did not differ in the latency to fall from the rotarodat different constant speeds (ANOVA ‘genotype’: vel 2: F(1,76)=0.63,p=0.42; vel 3: F(1,76)=1.54, p=0.21) or during the acceleration cycle(F(1,76)=1.87, p=0.17).

Furthermore, no differences were found between 8581- or R1- andvehicle-treated Ts65Dn or control mice in the latency to fall at thedifferent constant speeds (ANOVA ‘treatment’ vel 2: F(1,76)=0.08,p=0.92); vel 3: F(1,76)=1.42, p=0.24) or during the acceleration cycle(F(1,76)=1.40, p=0.25).

MANOVA revealed that there was no significant interaction of the factors‘genotype’ and ‘treatment’ in any of the conditions tested in therotarod (vel 2: F(1,76)=0.31, p=0.72; vel 3: F(1,76)=0.48, p=0.61;acceleration. F(1,76)=0.43, p=0.64).

Example 3. Spontaneous Activity: Actimetry

In this test the circadian variation of the animals' spontaneouslocomotor activity during a complete light-dark cycle of 24 hours wasevaluated. The apparatus is a device (Acti-system II, Panlab, Barcelona)that detects the changes produced in a magnetic field by the movement ofthe mice. It registers the movements of animals during a continuous 24hour cycle (12 hours of light and 12 hours of darkness).

FIGS. 3 and 4 show that Ts65Dn and control mice (ANOVA ‘genotype’ dark:F(1,76)=2.79, p=0.10; light: F(1,76)=2.24, p=0.14) under vehicle, R1 or8581 treatment (ANOVA ‘treatment’ dark: F(1,76)=2.20, p=0.12, light:F(1,76)=0.27, p=0.76; ANOVA ‘genotype×treatment’: dark: F(1,76)=0.79,p=0.45; light: F(1,76)=0.39, p=0.67) did not differ in the amount ofspontaneous activity performed in their home cage during the dark or thelight phase of the cycle.

Example 4. Open Field

Exploratory behavior and anxiety were assessed using a square-shapedopen field (55 cm×55 cm, surrounded by a 25-cm-tall fence), divided into25 equal squares. The animals were placed in the center of the field,and the number of vertical (rearing) activities and horizontal crossings(from square to square, subdivided into center vs. peripheral crossings)were scored in a single 5-min trial.

In the Open Field test, no significant differences were found in theactivity performed by mice of both genotypes in the center of the maze(ANOVA ‘genotype’: F(1,76)=2.77, p=0.10; FIG. 5) or in the number ofrearings (F(1,76)=0.01, p=0.90; FIG. 6). However, vehicle-treated Ts65Dnmice were hyperactive when compared to control mice under the sametreatment as shown by the increase in activity in the periphery (ANOVA‘genotype’: F(1,76)=15.86, p<0.001; FIG. 5), and in total activity(F(1,76)=17.39, p<0.001; FIG. 6).

MANOVA revealed that R1- or 8581-treatment did not significantly affecthorizontal (ANOVA ‘treatment’: periphery: F(1,76)=1.08, p=0.34; totalnumber of crossings: F(1,76)=1.27, p=0.28) or vertical (rearingsF(1,76)=1.75, p=0.18) activity in mice of either genotype. The fact thatchronic administration of these two compounds did not affect activity inthe center of the maze (ANOVA ‘treatment’: center: F(1,76)=2.42,p=0.096) suggests that these compounds did not produce an anxiogeniceffect in mice or either genotypes. No significant interaction was foundbetween ‘genotype’ and ‘treatment’ in horizontal activity (centerF(1,44)=0.64, p=0.71; periphery: F(1,76)=1.06, p=0.35; total:F(1,76)=1.00, p=0.37) but ANOVA revealed a significant effect of thesetwo factors on the number of rearings (F(1,76)=3.36, p=0.04).

Example 5. Exploratory Activity: Hole Board

The hole board is a wooden box (32×32×30 cm) with four holes. The flooris divided into nine 10 cm squares. In a single 5 minute trial thenumber of explorations, the time spent exploring each hole, and overallactivity in the apparatus were measured. A repetition index was alsocalculated (exploration of holes previously explored) as a function ofthe number of ABA alternations.

Table 4 shows the scores of R1-, 8581- and vehicle-treated Ts65Dn andcontrol mice in the Hole Board test. Ts65Dn mice under all treatmentsperformed a larger number of crossings than control mice. 8581 and R1treatment reduced this hyperactivity shown by Ts65Dn mice. Ts65Dn micealso showed an increase in the number of explorations performed underall treatment conditions. No differences were found in vertical activityin this maze among Ts65Dn and control mice under the differenttreatments. No significant differences were found between mice of bothgenotypes and treatments in the time they spent exploring the holes.Ts65Dn mice showed altered attention since they repeated a larger numberof times the exploration of recently explored holes (ABA index). After8581 (but not after R1) treatment, Ts65Dn mice ABA index was normalized.

Example 6. Spatial Learning: Morris Water Maze

To evaluate spatial learning the Morris Water Maze was used. Theapparatus was a circular tank of 110 cm in diameter, full of water(22-24° C.) made opaque by the addition of powdered milk. Inside thetank, a platform was hidden 1 cm below the water level. Animals weretested at the end of the treatment period in 12 consecutive dailysessions: 8 acquisition sessions (platform submerged), followed by 4cued sessions (platform visible). All trials were videotaped with acamera located 2 m above the water level. A SMART computerized trackingsystem (Panlab S.A., Barcelona, Spain) was used to analyze the mousetrajectories, measure escape latency, distance traveled, and swimmingspeed for each animal in each trial.

Training Sessions

In the acquisition sessions (S1-S8), the platform was hidden 1 cm belowthe water level. From one daily session to the next, the platform wasplaced in a different location (E, SW, center, and NW); each positionwas used once every four consecutive daily sessions. Each of the 8acquisition and 4 cued sessions (one session per day) consisted of fourpairs of trials, 30-45 min apart. For each trial pair, the mice wererandomly started from one of four positions (N, S, E, W), which was heldconstant for both trials. The first trial of a pair was terminated whenthe mouse located the platform or when 60 s had elapsed; the secondtrial commenced following a period of 20 s, during which the animal wasallowed to stay on the platform. Several fixed cues outside of the mazewere constantly visible from the pool.

Cued Sessions

During the cued sessions the platform was visible: the water level was 1cm below the platform, and its position was indicated with a flag. Eighttrials were performed during each session, following the sameexperimental procedure as in the acquisition sessions. As shown in FIG.7, all groups of mice learned the platform position throughout theacquisition sessions since they reduced the latency to reach theplatform (ANOVA ‘session’: F(7,65)=26.8, p<0.001).

Ts65Dn mice showed a pronounced learning deficit in the MWM (ANOVA‘genotype’: F(1,65)=39.26, p<0.001; FIG. 8A), but the difference betweenTs65Dn and control learning curves was reduced after 8581 (ANOVA‘genotype’: F(1,18)=4.69, p<0.05; FIG. 8B) and after R1 treatment (ANOVA‘genotype’: F(1,26)=13.57, p<0.01).

As shown in FIG. 9A, 8581-treatment significantly improved Ts65Dn miceperformance (ANOVA ‘treatment’: F(1,24)=32.43, p<0.001). Chronic R1treatment also improved Ts65Dn mice cognition (F(1,24)=9.2, p<0.01; FIG.9C). 8581 (FIG. 9B) or R1 (FIG. 9D) did not significantly affect controlmice performance.

During the cued sessions (FIG. 10), vehicle-treated Ts65Dn mice showedan increased latency to reach the platform with respect to control mice(ANOVA ‘genotype’: F(1,46)=5.35, p=0.024). R1 and 8581 treatment reducedthe latency to reach the platform (ANOVA ‘treatment’: F(1,46)=6.52,p=0.003) in Ts65Dn but not in control mice (ANOVA ‘genotype×treatment’:F(1,46)=3.44, p=0.038)

Example 7. Long Term Potentiation (LTP)

The effect of chronic administration of 8581 and R1 on LTP was evaluatedin the Ts65Dn mouse model of Down syndrome. 8581, R1 (20 mg/kg p.o.) orvehicle was administered for six weeks. Mice were decapitated 1 hourafter the last administration and the brains were rapidly removed. Thehippocampi were dissected and 400-μm slices were cut with a tissuechopper. Slices were allowed to recover for at least 1 hour in aninterface chamber at RT with artificial cerebral spinal fluid (ACSF)containing (in mM): 120 NaCl, 3.5 KCl, 2.5 CaCl₂, 1.3 MgSO₄, 1.25NaH₂PO₄, 26 NaHCO₃ and 10 D-glucose, saturated with 95% O₂ and 5% CO₂.Field excitatory postsynaptic potentials (fEPSPs) were recorded from theCA1 stratum radiatum with a glass micropipette (1-4 MΩ) containing 2 MNaCl and evoked by stimulation of the Schaffer collaterals withinsulated bipolar platin/iridium electrodes >500 μm away from therecording electrode. The stimulus strength was adjusted to evoke fEPSPsequal to 50% of the relative maximum amplitude without superimposedpopulation spike. After stable baseline recordings (100 μspulse-duration, 0.033 Hz), long term potentiation (LTP) was induced byTBS (10 trains of 5 pulses at 100 Hz and intervals of 200 ms). Theduration of the stimulation pulses was doubled during the tetanus. After20 min of baseline recording, LTP was induced and recorded for 80 min ineach individual hippocampal slice. Signals from recording electrodeswere amplified and bandpass-filtered (1 Hz-1 kHz) and stored in acomputer using the Spike 2 program (Spike2, Cambridge Electronic Design,Cambridge, UK). For the analysis, fEPSP slopes were expressed as apercentage of the baseline values recorded. Results from several sliceswere expressed as mean±SEM. The statistical analysis was carried out byrepeated-measures (RM) MANOVA (‘time’×‘treatment’×‘genotype’). All theanalyses were done using SPSS for Windows version 18.0.

As shown in FIGS. 11 and 12, hippocampal slices of vehicle treatedTs65Dn mice displayed deficits in LTP. In contrast, the LTP induced inhippocampal slices of 8581 or R1 treated animals was not different fromthat induced in hippocampal slices of control mice (FIGS. 11 and 12respectively). This suggests that chronically treating Ts65Dn mice with8581 or R1 rescue the deficit in LTP probably by reducing the excessiveGABA-mediated inhibition observed in these animals.

Example 8. Rescued Neurogenesis

Alterations in hippocampal morphology such as reductions in granule celldensity and hippocampal neurogenesis have also been implicated in thelearning deficits shown by Ts65Dn mice. Spatial learning is known todepend on the functional integrity of the hippocampus, a structure thatplays a key role in information encoding and retrieving in the CNS. Wehave studied the population of newborn cells in the dentate girus (DG)by labelling proliferating cells with anti-Ki67, a marker of cellsundergoing the late G1 phase and phases G2 and M. We confirmed thathippocampal neurogenesis was reduced in these mice and showed thatchronic administration of 8581 completely restored the density ofproliferating cells in TS mice (p=0.033; FIG. 13). Also, neuronalsurvival of the cells that have undergone maturation was also normalizedin TS mice, as shown by the increase in DAPI+cells found in TS miceafter chronic 8581 administration (p=0.026; FIG. 14).

Therefore, this compound facilitates cell proliferation and survival ofneurons that have undergone maturation. Since both new-born neurons andmature neurons seem to be implicated in hippocampus-dependent learningand memory, the restoration of proliferation and of the density ofmature neurons is likely to be implicated in the cognitive-enhancingeffects of 8581 in TS mice.

In addition, we found that in TS mouse hippocampus there is anenhancement in the number of GABAergic synapses compared to controlanimals. Importantly chronic treatment with 8581 rescue this alterationas the number of GAD positive boutons decreased dramatically afterchronic treatment with this selective GABAA α5 NAM (p=0.017; FIG. 15).This treatment produced a non-significant tendency to increase thenumber of these synapses in CO mice.

Example 9. Spatial Learning of Nf1+/− Mice in the Morris Water Maze

1 week after handling, mice were trained with two consecutive trials perday for 8-9 days 30 minutes after i.p. injection of 8581 or vehicle. Ineach trial, mice were given 60 s to find the platform. After each trial,mice were put on the platform for 15 s. On the day of probe trial (Day3, Day 5, Day 7, and Day 9) 60 s of probe trial was performed aftertraining. On probe trial 1 (day 3), none of the groups tested learned tosearch specifically in the target quadrant; probe trials 3 and 4 (days 7and 9) showed that the Nf1/veh group was significantly impaired comparedto CO/veh (two-way ANOVA, quadrant×genotype interaction, F(3,51)=5.662,P<0.01). Importantly, 8581-treated Nf1+/− mice show comparableperformance to control animals on all the probe trials, suggesting that8581 rescued the spatial learning deficits of Nf1+/− mice. Two differentmeasures of spatial learning (% search in quadrant (FIG. 16) and averageproximity to the target platform (FIG. 17)) show similarly, that activepharmaceutical compounds used in present invention rescue spatiallearning deficits of Nf1+/− mice (CO/veh (n=10), Nf1/veh (n=9), CO/8581(n=10), and Nf1/8581 (n=11).

Effects of active pharmaceutical compounds used in present invention onthe performance of Nf1+/− mice were tested under conditions in which thespatial learning of the mutants was indistinguishable from controls(less extinction due to lower number of probe trials). Mean percentageof time spent in each quadrant during a probe trial in plotted in FIG.18. Average proximity to the target quadrant is plotted in FIG. 19.Results show that Nf1+/− mice treated with vehicle wereindistinguishable from control mice similarly treated. Comparisonsbetween % search on target quadrant and proximity to target quadrant didnot reveal any difference between groups. On probe trial 1 and 2 (days 5and 7), all groups searched selectively in the target quadrant (p<0.01),and there were no differences between groups.

Example 10. Fear Conditioning

Control mice (B16;129F1) were trained with a contextualfear-conditioning protocol using either one trial per day for one day(FIG. 20) or two consecutive days (FIG. 21). On the training day, 30minutes after i.p. injection of 8581 or vehicle, mice were placed intraining chamber. A foot shock (1 s, 0.4 mA) was delivered 40 s afterplacement. Conditioned response (percentage freezing time of the mice)was recorded 24 h after training by using automated procedures. Averagefreezing levels during the first 30 s of each training day and 24 hrafter the last training trial were plotted.

The dose response curve (0.3, 1.0 and 3.0 mg/kg) as shown in FIG. 20reveals a dose-dependent increase in contextual fear conditioning incontrol mice. 3 mg/kg 8581 significantly enhanced freezing at 24 hrafter training (p<0.05). As can be seen from FIG. 21, 1 mg/kg 8581 alsocaused a significant increase in contextual conditioning when mice weretrained for two consecutive days. Mice treated with the drug freezesignificantly more compared to control/vehicle group (F(1,18)=5.254,p=0.034).

Example 11. Rotarod

Control mice (B16;129F1) and Nf1+/− mice were treated with vehicle orwith 8581 (n=10 for each of the 4 groups). 30 minutes after i.p.injection of 8581 (1 mg/kg) or vehicle, mice were tested with a rotarodprotocol using accelerating speeds (4-40 rpm, maximal duration 300 s)for four trials with 30 min intertrial interval. FIG. 22 visualizesperformance of control mice in the Rotarod and FIG. 23 illustrates theperformance of Nf1+/− mice in the Rotarod. 8581 did not affect theperformance of either Nf1+/− mutant or control mice.

TABLE 1 Control Ts65Dn 8581 10 10 R1 16 15 Vehicle 13 13

TABLE 2a hKi hKi hKi hKi Com- α5 α1 α2 α3 hKi α1/ hKi α2/ hKi α3/ pound[nM] [nM] [nM] [nM] hKi α5 hKi α5 hKi α5 8580 9.1 43.9 25.4 26.2 5 3 38581 4.6 174.3 185.4 79.6 38 40 17 8582 16.5 256.5 246.3 105.8 16 15 68583 1.6 28.3 13.9 10.5 18 9 7 8584 2.5 125.3 181.0 116.7 50 72 47 85850.3 3.8 1.5 0.7 14 5 2 8586 2.1 15.3 23.7 14.0 7 12 7 8587 10.9 249.8141.4 105.8 23 13 10 8588 1.1 20.5 40.1 16.0 19 37 15 8589 6.1 225.9228.7 215.8 37 37 35 8590 0.5 2.1 3.7 3.9 4 7 8 8591 7.1 624.0 668.6515.4 88 94 73 O1 1.3 504.3 100.5 110.2 388 77 85 P1 29.9 Q1 15.1 633.0437.9 399.6 42 29 26 R1 4.7 984.6 501.5 489.1 209 107 104 S1 5.4 550.8209.3 220.2 102 39 41 T1 14.6 467.9 292.8 387.1 32 20 27 U1 0.3 45.8 7.27.5 153 24 25 V1 1.2 143.3 53.3 76.1 119 44 63 W1 0.4 34.6 10.7 17.7 8727 44

TABLE 2b Efficacy [%] Efficacy [%] Efficacy [%] Efficacy [%] GABA A GABAA GABA A GABA A Compound α5β3γ2 α1β2/3γ2 α2β3γ2 α3β3γ2 8580 −35 8581 −46−1 −11 −1 8582 −37 −21 8585 −41 −37 8586 −33 −5 +2 +7 8587 −42 −28 8591−31 +4 −7 +23 O1 −50 −19 −34 −43 Q1 −35 −11 −18 −17 R1 −39 −4 −6 −2 T1−31 −3 +5 +9 U1 −33 −15 −34 −21 V1 −33 0 +9 +16

TABLE 3 MANOVA ‘genotype x treatment’ treat- gen x Vehicle 8581 R1genotype ment F treat- Ts65Dn control Ts65Dn control Ts65Dn control F(1.76) (2.76) ment Vision 8.23 ± 8.54 ± 7.67 ± 7.64 ± 9.19 ± 8.40 ±0.09, 1.74, 0.53, 0.60 0.57 0.88 0.47 0.54 0.52 p = 0.76 p = 0.18 p =0.58 Auditory 1.15 ± 1.31 ± 1.33 ± 1.00 ± 1.31 ± 1.00 ± 1.07, 0.10,0.92, startle 0.19 0.24 0.17 0.13 0.18 0.20 p = 0.30 p = 0.90 p = 0.40Righting 3.00 ± 3.00± 3.00 ± 3.00 ± 3.00 ± 3.00 ± reflex 0.00 0.00 0.000.00 0.00 0.0 Grip strength 1.62 ± 2.00 ± 2.00 ± 1.55 ± 1.75 ± 2.2 ±0.74, 0.63, 2.33, 0.24 0.28 0.24 0.21 0.19 0.22 p = 0.39 p = 0.53 p =0.10 Equilibrium 3.08 ± 2.4 ± 3.55 ± 2.8 ± 3.19 ± 2.53 ± 7.58, 0.80,0.02, wooden bar 0.37 0.24 0.38 0.38 0.31 0.24  p = 0.008 p = 0.45 p =0.97 Latency to 20.0 ± 19.8 ± 17.77 ± 20.00 ± 20.00 ± 20.00 ± 1.62,1.62, 1.89, fall wooden 0.0 0.19 2.22 0.00 0.0 0.0 p = 0.20 p = 0.20 p =0.15 bar Equilibrium 2.31 ± 1.38 ± 1.00 ± 1.36 ± 1.13 ± 0.80 ± 0.50,2.09, 0.80, aluminium 0.54 0.43 0.44 0.45 0.33 0.34 p = 0.48 p = 0.13 p= 0.45 bar Latency to 15.15 ± 13.42 ± 12.11 ± 14.73 ± 13.71 ± 11.16 ±0.10, 0.64, 1.39, fall 1.55 1.33 2.07 1.27 1.32 1.57 p = 0.74 p = 0.52 p= 0.25 aluminium bar Prehensile 2.92 ± 2.69 ± 2.67 ± 2.45 ± 2.75 ± 2.53± 1.63, 0.67, 0.00, reflex 0.83 0.17 0.24 0.31 0.17 0.22 p = 0.20 p =0.51 p = 1.00 Traction 2.67 ± 2.31 ± 1.67 ± 1.82 ± 2.50 ± 1.80 ± 0.390.72, 0.25, capacity 0.62 0.63 0.62 0.55 0.57 0.45 p = 0.53 p = 0.48 p =0.77 Number of 3.67 ± 3.62 ± 3.33 ± 2.64 ± 3.19 ± 2.73 ± 1.76, 1.36,0.71, crossings coat 0.34 0.96 0.50 0.43 0.73 0.36 p = 0.18 p = 0.26 p =0.49 hanging Latency 32.42 ± 36.00 ± 35.44 ± 46.45 ± 37.37 ± 30.93 ±0.34, 0.85, 1.22, arrival coat 6.16 5.62 5.76 5.79 4.91 5.16 p = 0.55 p= 0.42 p = 0.29 hanging

TABLE 4 MANOVA ‘genotype x treatment’ treat- gen x Vehicle 8581 R1genotype ment treat- s65Dn control Ts65Dn control Ts65Dn control F(1.76)(2.76) ment Crossings 127.15** ± 79.00 ± 106.11 ± 73.27 ± 93.19 ± 72.13± 23.55,  3.27, 1.42, 11.86 5.24 9.80 8.31 7.50 7.05 p < 0.001 p = 0.04p = 0.24 Rearings 13.77 ± 11.54 ± 20.67 ± 17.73 ± 13.19 ± 13.86 ± 0.41,2.77, 0.24, 2.55 1.98 5.69 3.06 2.20 2.01 p = 0.52  p = 0.07 p = 0.78Number of 22.62 ± 18.15 ± 18.22 ± 14.82 ± 20.50 ± 16.13 ± 6.18, 1.71,0.03, Head- 2.21 1.84 1.96 2.00 1.95 1.71 p = 0.015 p = 0.18 p = 0.96dippings Time 37.88 ± 33.92 ± 26.28 ± 30.43 ± 37.58 ± 28.28 ± 0.36,0.68, 0.58, exploring 7.55 6.63 4.17 6.83 5.07 4.62 p = 0.54  p = 0.50 p= 0.56 holes ABA index 5.23 ± 3.23 ± 3.89 ± 2.64 ± 4.25 ± 4.06 ± 4.21,1.10, 1.02, 0.60 0.53 0.72 0.86 0.72 0.56 p = 0.044 p = 0.33 p = 0.36

TABLE 5 Dose (mg/kg) 0.1 1 Genotype Nf1+/− control Nf1+/− control 0.5 h10.97 ± 1.73 9.45 ± 3.05  192 ± 35 271 ± 1  3 h <0.5 <0.5 8.945 ± 0.511.89 ± 2.51 7 h <0.5 <0.5 <0.5 <0.5 24 h <0.5 <0.5 <0.5 <0.5

1. A method of treating or delaying the progression of CNS conditionsrelated to excessive GABAergic inhibition in the cortex and hippocampuscomprising administering to a subject having such condition atherapeutically effective amount of a GABA A α5 negative allostericmodulator of formula (I) or formula (II)

wherein R¹ is hydrogen, halo, alkyl, haloalkyl, or cyano; R² ishydrogen, halo, alkyl, haloalkyl, or cyano; R³ is hydrogen, alkyl, orheterocycloalkylalkyl, wherein heterocycloalkylalkyl is optionallysubstituted with one or more hydroxy, oxo, alkyl, alkoxy, haloalkyl,hydroxyalkyl, halo, or cyano; R⁴ is aryl or heteroaryl, each optionallysubstituted by one, two or three halo; R⁵ is hydrogen, alkyl, haloalkylor hydroxyalkyl; R⁶ is —C(O)—NR⁷R⁸ R⁷ is hydrogen; R⁸ is alkyl; or R⁷and R⁸ together with the nitrogen to which they are bound form aheterocycloalkyl, or a heteroaryl, each optionally substituted with oneor more hydroxy, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, halo, orcyano; or a pharmaceutically acceptable salt thereof.
 2. The method ofclaim 1, wherein the excessive GABAergic inhibition in the cortex andhippocampus is caused by neurodevelopmental defects.
 3. The method ofclaim 1, wherein the CNS conditions are caused by neurodevelopmentaldefects which result in excessive GABAergic inhibition in the cortex andhippocampus.
 4. The method of claim 1, wherein said CNS condition isselected from cognitive deficits in Down Syndrome, cognitive deficits inautism and cognitive deficits in neurofibromatosis type I.
 5. The methodof claim 4, wherein said CNS condition is cognitive deficits in DownSyndrome.
 6. The method of claim 4, wherein said CNS condition iscognitive deficits in autism.
 7. The method of claim 4, wherein said CNScondition is cognitive deficits in neurofibromatosis type I.
 8. Themethod of claim 1, wherein said CNS condition is cognitive deficitsafter stroke.
 9. The method of claim 1, wherein said GABA A α5 negativeallosteric modulator binds to human GABA A α5β3γ2 receptor subtype witha binding selectivity of a factor of 10 or more as compared to bindingaffinities to human GABA A α1β2/3γ2, α2β3γ2 and α3β3γ2 receptorsubtypes.
 10. The method of claim 1, wherein said GABA A α5 negativeallosteric modulator exhibits a functional selectivity by acting asinverse agonist at human GABA A α5β3γ2 receptor subtype by reducing theeffect of GABA by more than 30% and in addition affecting the effect ofGABA at human GABA A α1β2/3γ2, α2β3γ2 and α3β3γ2 receptor subtypes byless than 15%.
 11. The method of claim 1, wherein said GABA A α5negative allosteric modulator is selected from a compound of formula (I)or a pharmaceutically acceptable salt thereof.
 12. The method of claim11, wherein R¹ is hydrogen, halo, haloalkyl, or cyano; R² is halo, orhaloalkyl; R³ is hydrogen, alkyl, or heterocycloalkylalkyl substitutedwith one oxof.
 13. The method of claim 12, wherein said GABA A α5negative allosteric modulator is selected from3-Fluoro-10-fluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;3-Bromo-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;3-Cyano-10-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;10-Difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;3-Chloro-10-fluoromethyl-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;10-Chloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;3,10-Dichloro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;10-Chloro-3-cyano-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;10-Chloro-3-difluoromethyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;3-Bromo-10-chloro-6-methyl-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;10-Bromo-3-fluoro-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;and3-Bromo-10-methyl-6-(2-oxo-pyrrolidin-1-ylmethyl)-9H-imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]benzodiazepine;or a pharmaceutically acceptable salt thereof.
 14. The method of claim1, wherein said GABA A α5 negative allosteric modulator is selected froma compound of formula (II) or a pharmaceutically acceptable saltthereof.
 15. The method of claim 14, wherein R⁴ is aryl or heteroaryl,each optionally substituted by one halo; R⁵ is alkyl; R⁶ is C(O)—NR⁷R⁸;R⁷ is hydrogen and R⁸ is alkyl; or R⁷ and R⁸ together with the nitrogento which they are bound form a heterocycloalkyl optionally substitutedwith one or two oxo, or form a heteroaryl.
 16. The method of claim 15,wherein said GABA A α5 negative allosteric modulator is selected from:N-Isopropyl-6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-nicotinamide;(5,6-Dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-[6-(5-methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-methanone;[6-(5-Methyl-3-phenyl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-methanone;(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;{6-[3-(4-Chloro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-morpholin-4-yl-methanone;[6-(5-Methyl-3-pyridin-2-yl-isoxazol-4-ylmethoxy)-pyridin-3-yl]-morpholin-4-yl-methanone;6-[3-(5-Fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-isopropyl-nicotinamide;(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(5-fluoro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanone;and{6-[3-(5-Chloro-pyridin-2-yl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-thiomorpholin-4-yl-methanone;or a pharmaceutically acceptable salt thereof.
 17. The method of claim16, wherein said GABA A α5 negative allosteric modulator is(1,1-Dioxo-1,6-thiomorpholin-4-yl)-{6-[3-(4-fluoro-phenyl)-5-methyl-isoxazol-4-ylmethoxy]-pyridin-3-yl}-methanoneor a pharmaceutically acceptable salt thereof.
 18. The method of claim1, wherein said GABA A α5 negative allosteric modulator is usedseparately, sequentially or simultaneously in combination with a secondactive pharmaceutical compound.